Method of detecting pathogenic legionella strains

ABSTRACT

The invention relates to a kit for detecting pathogenic  Legionella pneumophila  strains by hybridizing genomic DNA of a sample suspected to contain  Legionella  to two or five specific sequence markers, as identified by MARKER NO. 1 through MARKER NO. 5 or homologues thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 12/521,119, having an international filing date of 28 Dec. 2007, now allowed, which is the National Stage Application of PCT/NL2007/000332, filed 28 Dec. 2007, which claims benefit of European application No. 06077343.9 filed 29 Dec. 2006. The contents of the above patent applications are incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 313632007010SeqList.txt, date recorded: Apr. 2, 2013, size: 21,375 byes).

FIELD OF THE INVENTION

The invention relates to a method of typing Legionella pneumophila strains, and in particular to a method of identifying a Legionella pneumophila strain as being either pathogenic or non-pathogenic. The invention further relates to markers for such an assay and a kit of parts comprising an array with said markers and reference materials for performing a method of the invention.

BACKGROUND OF THE INVENTION

Legionnaires' disease is an acute pneumonic illness caused by Gram-negative bacilli of the genus Legionella, the most common of which is Legionella pneumophila.

Legionnaires' disease is initiated by inhalation, and probably microaspiration, of Legionella bacteria into the lungs. Although Legionella bacteria are ubiquitous in our environment, they rarely cause disease. A number of factors must occur simultaneously before legionnaires' disease is possible. These factors include the presence of virulent strains in an environmental site; a means for dissemination of the bacteria, such as by aerosolization; and proper environmental conditions allowing the survival and inhalation of an infectious dose of the bacteria by a susceptible host.

Water contaminated with a sufficient concentration of virulent Legionella bacteria can be aerosolized by water-cooled heat rejection devices such as air conditioning cooling towers, whirlpool spas, shower heads, water misters, and the like. Once the bacteria enter the lung, they are phagocytosed by alveolar macrophages and then grow intracellularly. The Legionella bacteria produce virulence factors that enhance phagocytosis. After sufficient intracellular growth, the bacteria kill the macrophage, escape into the extracellular environment and are then rephagocytosed by other macrophages. Within a few days after initial infection, the bacterial concentration in the lung increases considerably. The resulting infiltration of the alveoli by neutrophils, additional macrophages and erythrocytes results in capillary leakage and edema and the chemokines and cytokines released by the macrophages help trigger a severe inflammatory response, which may be fatal.

More than 49 different Legionella species, encompassing 70 serogroups, have been described since its first discovery in 1977, 20 of which have been reported to infect humans. L. pneumophila contains at least 16 different serogroups. L. pneumophila serogroup 1 caused the 1976 Philadelphia outbreak and is the cause of 70% to 90% of all cases of legionnaires' disease. In the major outbreaks such as those originating in Bovenkarspel, The Netherlands in 1999, in Barrow-in-Furness in Cumbria, England in 2002 and near Harnes in Pas-de-Calais, France in 2003/2004, serogroup 1 strains could be detected in the majority of the patients.

L. pneumophila serogroup 1 can be further divided into multiple subtypes using a variety of serologic, other phenotypic and genetic methods. One particular subtype of L. pneumophila serogroup 1 causes the majority of cases of legionnaires' disease due to L. pneumophila, and 85% of the cases due to L. pneumophila serogroup 1; this subtype is distinguished by its reactivity with a particular monoclonal antibody, and it is variously termed Pontiac, the Joly monoclonal type 2 (MAb2), or the Dresden monoclonal type 3/1 (MAb 3/1) monoclonal subtype.

Most clinical microbiology laboratories are capable of identifying Legionella bacteria to the genus level by detection of their typical colony morphology, Gram stain appearance, and various other standard microbiology identification techniques. Identification of L. pneumophila serogroup 1, the most common clinical isolate can be accomplished by sophisticated clinical microbiology laboratories using relative simple serologic testing. However, it should be stressed that serogroup 1 strains are not the only virulent serotypes. Identification of other L. pneumophila serogroups, and other Legionella species, is often much more difficult. This is because these bacteria are relatively inert in the use of commonly tested biochemical substrates, and they require sophisticated phenotypic, serologic and molecular testing. Reference laboratory-based phenotypic testing of bacteria, including determination of fatty-acids and ubiquinones and protein electrophoresis, can often be used to identify the bacteria. Definitive identification is based on both immunologic detection of surface antigens and bacterial DNA sequencing. Also, DNA typing by pulsed field electrophoresis of DNA restriction fragments is useful for identification purposes.

Diagnosis of the infecting agent from clinical material (such as sputum or bronchoalveolar lavage fluid) on selective media is currently the most reliable means of diagnosis. However, cultivation is slow. Direct fluorescent antibody (DFA) stains for the visualization of Legionella species in clinical specimens are commercially available for a limited number of species. Assays for the detection of L. pneumophila serogroup 1 antigen in urine have a sensitivity of 70% and a specificity of nearly 100%. Also, an immunochromatographic assay for the rapid qualitative detection of L. pneumophila serogroup 1 antigen (Legionella NOW; Binax, Portland, Me.) in urine specimens has become available that uses rabbit anti-L. pneumophila serogroup 1 antibody as the capture component and rabbit anti-L. pneumophila serogroup 1 antibody conjugated to colloidal gold as the detection component. The assay provides a test result in 15 min and is intended to aid in the presumptive diagnosis of Legionnaires' disease caused by L. pneumophila serogroup 1 in conjunction with culture and other methods. Preliminary performance data for the immunochromatographic assay report a sensitivity of 95% and a specificity of 95%. Thus, so far, commercially available tests for Legionella urinary antigen detect only L. pneumophila serogroup 1, while the specificity of the assays cannot prevent the occurrence of false positive and false negative reactions.

DNA probe techniques, which produce fewer false positive reactions then immunological detection methods, may be used to detect the presence of one or more multiple Legionella species. However, a drawback of such DNA methods is that they cannot differentiate between virulent and non-virulent strains, presumably because the virulence trait is multi-genic.

Following severe outbreaks, many national authorities have implemented legislation and water quality standards for water supplies and/or codes of practice for management and operation of cooling towers and warm water storage facilities. Such standards and codes require frequent monitoring of drinking water distribution systems and swimming-pool water facilities, and upon exceeding a certain number of Legionella bacteria per liter, rigorous measures are taken, such as closure and evacuation of hotels, sports facilities or nursing homes. Most experts however, consider that a large number of Legionella bacteria detected are harmless and non-virulent. However, there is at present no assay system available to distinguish between virulent and non-virulent strains and it is difficult to avoid the costs involved with false-alarm Legionella detection. The availability of a test that is capable of reliably detecting pathogenic Legionella strains would be highly favorable.

It is an object of the present invention to provide for a method capable of distinguishing between clinically relevant and environmental strains of L. pneumophila. It is a further object of the present invention to provide a method of detecting pathogenic Legionella strains including, but not limited to serogroup 1 strains.

SUMMARY OF THE INVENTION

The present inventors have now found a method for distinguishing between clinical or pathogenic strains and environmental or non-pathogenic Legionella strains. The method involves an hybridization assay with specific genetic markers.

In one embodiment of the present invention, the group of molecular markers comprises the two markers MARKER NO. 1 and MARKER NO. 2, having sequences 1 a, 1 b and 2a and 2b, respectively, or sequences which are highly homologous to those.

In a preferred embodiment of the method of the present invention, the group of molecular markers comprises the five markers MARKER NO. 1, MARKER NO. 2, MARKER NO. 3, MARKER NO. 4 and MARKER NO. 5, having the sequences as presented in the sequence listing or homologues thereof.

In a further embodiment of the method of the present invention, the sample nucleic acid is derived from a pure culture of Legionella pneumophila. Alternatively, the sample can be derived from any medium which is suspected of Legionella infestation, such as aqueous samples from water pipes, surface water, drink water, showers, baths, fountains, etc.

A kit of parts, said kit comprising an array as defined herein together with reference nucleic acids as defined herein. Preferably, said kit also comprises software to calculate the result of the test.

DETAILED DESCRIPTION OF THE INVENTION

The term “Legionella pneumophila strain” as used herein refers to the descendants of a single isolate of Legionella pneumophila cells in pure culture and to a taxonomic level of said pure culture below the level of the species Legionella pneumophila.

The term “pathogenic” in relation to a Legionella strain refers to a microorganism capable of causing disease (including infection) or morbid symptoms in humans or other animal hosts.

The term “array” refers to an array of individual fragments of DNA or oligonucleotides that are bound to a substrate such as a microscope slide. The purpose of an array experiment is to determine the amount of matching nucleic acid fragments in a particular sample. The target nucleic acid fragments are extracted from the cells or tissues of interest, optionally converted to DNA, and labeled. They are then hybridized to all the DNA or oligonucleotide spots on the array. Matches (i.e. the spots where hybridization has taken place) are identified using antibody detection, optionally combined with precipitation, chromatography, colorimetry, and/or phosphorescent or fluorescent imaging. The data resulting from an array experiment are a list of measurements of spot intensities. A preferred array of the invention is an array which has less than 1000, preferably less than 100, more preferably less than 50 and most preferably less than 10 spots, and which contains at least the DNA sequences of MARKER NO. 1 and MARKER NO. 2. Another preferred array of the invention is an array which has less than 1000, preferably less than 100, more preferably less than 50 and most preferably less than 10 spots, and which contains at least the five sequences as presented in MARKER NO. 1 through MARKER NO. 5.

The term “nucleic acid” as used herein, is interchangeable with the term “polynucleotide”, and refers to a nucleotide multimer or polymeric form of nucleotides having any number of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g. PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) and can be either double- or single-stranded. A polynucleotide can hybridize with other polynucleotides in a sequence specific manner, e.g. can participate in Watson-Crick base pairing interactions. The term also includes modified, for example by methylation and/or by capping, and unmodified forms of the polynucleotide

The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

The term “oligonucleotide” refers to a short sequence of nucleotide monomers (usually 6 to 100 nucleotides) joined by phosphorous linkages (e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), or non-phosphorous linkages (e.g., peptide, sulfamate and others). An oligonucleotide may contain modified nucleotides having modified bases (e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2′-O-methyl ribosyl, 2′-O-methoxyethyl ribosyl, 2′-fluoro ribosyl, 2′-amino ribosyl, and the like). Oligonucleotides may be naturally-occurring or synthetic molecules of double- and single-stranded DNA and double- and single-stranded RNA with circular, branched or linear shapes and optionally including domains capable of forming secondary structures (e.g., stem-loop, pseudo knots and kissing loop structures).

The term “nucleotide sequence homology” as used herein denotes the presence of homology between two polynucleotides. Polynucleotides have “homologous” sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence. Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. The “percentage of sequence homology” for polynucleotides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence homology may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence homology. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990; Altschul et al., 1997) and ClustalW programs, both available on the internet. Other suitable programs include, but are not limited to, GAP, BestFit, PlotSimilarity, and FASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, Wis., USA) (Devereux et al., 1984).

As used herein, “highly homologous” means that two nucleic acid sequences have at least about 85%, preferably at least 90%, more preferably at least 92%, even more preferably at least 95%, and most preferably at least 98%, sequence complementarity to each other. This means that primers and probes must exhibit sufficient complementarity to their template and target nucleic acid, respectively, to hybridise under stringent conditions. Therefore, the primer and probe sequences need not reflect the exact complementary sequence of the binding region on the template and degenerate primers can be used. For example, a non-complementary nucleotide fragment may be attached to the 5′-end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer has sufficient complementarity with the sequence of one of the strands to be amplified to hybridize therewith, and to thereby form a duplex structure which can be extended by the polymerising means. The non-complementary nucleotide sequences of the primers may include restriction enzyme sites. Appending a restriction enzyme site to the end(s) of the target sequence would be particularly helpful for cloning of the target sequence. A substantially complementary primer sequence is one that has sufficient sequence complementarity to the amplification template to result in primer binding and second-strand synthesis. The skilled person is familiar with the requirements of primers to have sufficient sequence complementarity to the amplification template.

The term “hybrid” in the context of nucleic acids refers to a double-stranded nucleic acid molecule, or duplex, formed by hydrogen bonding between complementary nucleotide bases. The terms “hybridise” or “anneal” refer to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary bases.

The term “probe” refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.

The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerisation such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerisation. The exact lengths of the primers will depend on many factors, including temperature and composition (A/T en G/C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

It will be understood that “primer”, as used herein, may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified. Hence, a “primer” includes a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing.

The oligonucleotide primers may be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods may include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in e.g. U.S. Pat. No. 4,458,066. The primers may be labelled, if desired, by incorporating means detectable by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical means.

Template-dependent extension of the oligonucleotide primer(s) is catalysed by a polymerising agent in the presence of adequate amounts of the four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP and dTTP, i.e. dNTPs) or analogues, in a reaction medium which is comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyse primer- and template-dependent DNA synthesis. Known DNA polymerases include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, and Taq DNA polymerase. The reaction conditions for catalysing DNA synthesis with these DNA polymerases are known in the art.

The products of the synthesis are duplex molecules consisting of the template strands and the primer extension strands, which include the target sequence. These products, in turn, serve as template for another round of replication. In the second round of replication, the primer extension strand of the first cycle is annealed with its complementary primer; synthesis yields a “short” product which is bound on both the 5′- and the 3′-ends by primer sequences or their complements. Repeated cycles of denaturation, primer annealing, and extension result in the exponential accumulation of the target region defined by the primers. Sufficient cycles are run to achieve the desired amount of polynucleotide containing the target region of nucleic acid. The desired amount may vary, and is determined by the function which the product polynucleotide is to serve.

The PCR method is well described in handbooks and known to the skilled person.

After amplification by PCR, the target polynucleotides may be detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions will be used. If some mismatching is expected, for example if variant strains are expected with the result that the probe will not be completely complementary, the stringency of hybridization may be lessened. However, conditions are chosen which rule out non-specific/adventitious binding. Conditions which affect hybridization, and which select against non-specific binding are known in the art, and are described in, for example, Sambrook et al., (2001). Generally, lower salt concentration and higher temperature increase the stringency of binding. For example, it is usually considered that stringent conditions are incubations in solutions which contain approximately 0.1×SSC, 0.1% SDS, at about 65° C. incubation/wash temperature, and moderately stringent conditions are incubations in solutions which contain approximately 1-2×SSC, 0.1% SDS and about 50°-65° C. incubation/wash temperature. Low stringency conditions are 2×SSC and about 30°-50° C.

The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures are well known in the art and are described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001.

The term “fragmented genomic DNA” refers to pieces of DNA of the genome of a cell that are the result of the partial physical, chemical or biological break-up of the lengthy DNA into discrete fragments of shorter length.

The term “hybridization pattern” refers to the list of measurements of spot intensities obtained after hybridizing the array with a target nucleic acid.

The term “nucleotide” is used to denote a deoxyribonucleoside, a ribonucleoside, or a 2′-O-substituted ribonucleoside residue

The term “molecular marker” generally refers to markers identifying variation at the level of DNA and is herein used to refer to a mutation (of any type) or nucleotide sequence which has a scorable or selectable relation with either the pathogenic or non-pathogenic phenotype (and hence can “mark” a region of the chromosome).

It now has been found, from a series of experiments comparing pathogenic and non-pathogenic Legionella strains, that a good discrimination can be achieved by detecting the presence of at least two marker sequences. Said markers are indicated as the sequences of MARKER NO. 1: sequences 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2) and MARKER NO. 2: sequences 2a (SEQ ID NO: 3) and 2b (SEQ ID NO: 4). Sequences 1a and 1b form part of a larger DNA fragment, which in total can be said to be functioning as the MARKER NO. 1 in the present invention. For detection of the presence of said MARKER NO. 1, it is sufficient if either or both of the sequences 1a and 1b are detected. A similar indication is also used for sequences 2a and 2b, which can be said to be part of the MARKER NO. 2. In a typical experiment said markers are present on a hybridization array and DNA of a Legionella strain to be classified as pathogenic or non-pathogenic is allowed to hybridize with said markers. Thus, for detecting the presence of MARKER NO. 1 and MARKER NO. 2, both sequence 1a and/or 1b and sequence 2a and/or 2b can be present on said array.

In another embodiment of the present invention, the array comprises at least the five MARKERS NOS. 1-5. The sequences of MARKERS NOS. 1 and 2 are discussed above. The sequences of MARKER NO. 3 are sequence 3a (SEQ ID NO: 5) and sequence 3b (SEQ ID NO: 6). MARKER NO. 4 is characterized by 5 sequences: 4a (SEQ ID NO: 7), 4b (SEQ ID NO: 8), 4c (SEQ ID NO: 9), 4d (SEQ ID NO: 10) and 4e (SEQ ID NO: 11). MARKER NO. 5 again is characterized by two sequences, 5a (SEQ ID NO: 12) and 5b (SEQ ID NO: 13). As indicated above, this does not necessarily mean that all of the sequences of a specific marker need to be present on the array: the assay on pathogenicity can equally well been performed with sequences, which are parts of the MARKERS NOS. 1-5. It should, however, be understood, that a minimal length is necessary for obtaining a correct and distinctive hybridization with any sample nucleic acid. Thus, the length of these part(s) of the sequences should range from about 50 to about 2000 nucleotides, i.e. the sequences can be shorter or longer than those presented in the sequence listing, but for a sufficient hybridization to occur they should be at least 50 nucleotides long. In other words: the presence of marker no. 4 can be detected by performing an assay with either sequence 4a, 4b, 4c, 4d and 4e, or with any combination of those sequences. Longer sequences than those depicted can be obtained by hybridizing the genome from a Legionella species and, if hybridization occurs with the presented sequence, excise a larger part of the sequence from the genomic DNA.

Further, hybridization allows for some mismatches between the sequence on the array and the nucleotide sequence of the sample. Therefore, any nucleotide sequence or part thereof (as defined above) which has a homology of at least 85%, preferably at least 90%, more preferably at least 92%, more preferably at least 95%, even more preferably at least 99% with the sequences of SEQ ID NO: 1 through SEQ ID NO: 13 is applicable in and intended within the scope of the present invention.

In the current invention the term “homologues” is used to indicate both parts of the sequences of the invention of at least 50, but preferably at least 100, more preferably at least 200, more preferably at least 500 and most preferably at least 1000 nucleotides, and sequences, which have a degree of homology of at least 85%, preferably at least 90%, more preferably at least 92%, more preferably at least 95%, even more preferably at least 99% with the sequences of SEQ ID NO: 1 through SEQ ID NO: 13.

The above discussed markers, or fragments thereof (such as the sequences of SEQ ID NO: 1 through SEQ ID NO: 13) may be spotted on a surface to provide for a DNA micro-array. In order to facilitate coupling of the fragments, the surface of the array (e.g. the slide, the surface of which may i.a. be glass, gold, etc.) may be modified. Spotting may occur by any method available, for instance by using ElectroSpray Ionization (ESI) micro-array printing. After spotting of the markers or fragments thereof, the slide surfaces may be blocked to prevent further attachment of nucleic acids, e.g. by treatment with boro-anhydride in case of formaldehyde modified glass-slide surfaces.

To facilitate detection of successful hybridization, the gDNA is suitably labeled, preferably with a compound which is uniquely detectable by an antibody. Such a compound can for instance be biotin. Labeling of nucleotides with biotin and subsequent detection through antibodies specific for biotin, has been sufficiently described in the literature and will be routine experimentation for a person skilled in the art. Alternatively, the nucleotides are fluorescently labeled (e.g. by using Cy™ labels [Amersham Pharmacia Biotech]). Fluorescent labeling kits are commercially available from various manufacturers. In order to be able to judge the signals caused by the hybridization of the marker sequences with sample nucleic acid, preferably the array also comprises reference nucleotide sequences, which can serve as positive and negative controls. As negative controls, sequences should be used of about the same length as the average length of the markers, but which will not hybridize with any nucleotide sequence in the sample, i.e. sequences, which do not occur in Legionella. As positive control, a sequence should be used which will be present in (nearly) all samples to be tested. For this purpose preferably the Legionella 30S ribosomal protein S21 (Cazalet, C., Rusniok, C., Bruggemann, H., Zidane, N., Magnier, A., Ma, L., Tichit, M., Jarraud, S., Bouchier, C., Vandenesch, F., Kunst, F., Etienne, J., Glaser, P. and Buchrieser, C. Nat. Genet. 36 (11), 1165-1173 (2004)) or a conserved sequence of one of the housekeeping enzymes should be used.

The average size of sample nucleic acid has an effect on the signal distribution on the array. Larger sample molecules comprise more information and are thus more likely to find a suitable hybridization partner in more of the spots. Reducing the average size of the sample nucleic acid can reduce this phenomenon. On the other hand, when the sample nucleic acid is too small, the nucleic acid fragments in the sample contain too little genetic information and also find suitable hybridization partners in many spots. The average size of the fragments in the sample nucleic acid is preferably between about 30 and 3000 nucleotides. More preferably, the average size of the fragments in the sample nucleic acid comprises a size of between about 50 and 1000 nucleotides, more preferably between about 100 and 500 nucleotides.

In a method for detecting a pathogenic L. pneumophila strain, the sample nucleic acid may represent the whole or a part of the sample genome. Preferably, at least those parts of the genome are present in which the presence of molecular markers as defined herein is to be detected. This can be achieved by randomly digestion of the genomic DNA of the sample to fragments of about 1.5 kb or by physically fragmenting the DNA (e.g. by shearing) to form fragments of about that size. In a preferred embodiment a PCR amplification step is performed on either the intact genomic DNA or on the fragmented DNA with primers that specifically cause amplification of one or more of the sequences of the invention. Alternatively, the DNA can be randomly (primer) labeled using Klenow DNA polymerase (BioPrime kit Invitrogen) according to the manufacturer's instructions.

The fragmented sample DNA is then brought into contact with the array of the invention, which contains at least the two marker sequences MARKER NO. 1 and MARKER NO. 2 or fragments thereof (such as the sequences of SEQ ID NO: 1 through SEQ ID NO: 4), or homologues of said markers or fragments thereof, or, in another embodiment, the five marker sequences of the inventions or fragments thereof (such as the sequences of SEQ ID NO: 1 through SEQ ID NO: 13), or homologues of said markers or fragments thereof. Hybridization of the sample nucleotides with the marker sequences and the hybridization signals with optional positive and negative control sequences is then determined. Then, the diagnosis whether or not the sample contained a pathogenic Legionella strain is done according to the following logic rule for the assay with only MARKER NO. 1 and MARKER NO. 2. In said rule the value for MARKER NO. 1 or 2 is 1 if a positive hybridization signal is obtained with respect to said sequence, and it is 0 if no positive hybridization signal is detected.

If (MARKER NO. 1>MARKER NO. 2) then Pathogenic else Environmental

For the assay with all five sequences, the following set of 7 rules is applicable. If 4 or more of these rules are TRUE if the stochastic 0 or 1 value for each hybridization is entered, then the sample can be classified as pathogenic.

If (MARKER NO. 2<MARKER NO. 1) then [not(MARKER NO. 2=MARKER NO. 3)] else [(MARKER NO. 2=MARKER NO. 4) and (MARKER NO. 5=MARKER NO. 3)]

If (MARKER NO. 2<MARKER NO. 3) then [not(MARKER NO. 1<MARKER NO. 4)] else [(MARKER NO. 5>MARKER NO. 2) nor (MARKER NO. 5<MARKER NO. 4)]

If (MARKER NO. 2<MARKER NO. 3) then [not(MARKER NO. 4<MARKER NO. 1)] else [(MARKER NO. 3<MARKER NO. 5) nor (MARKER NO. 4>MARKER NO. 5)]

If (MARKER NO. 1=MARKER NO. 5) then [(MARKER NO. 4<=MARKER NO. 1)] else [(MARKER NO. 1<MARKER NO. 4) nor (MARKER NO. 3=MARKER NO. 2)]

If (MARKER NO. 1=MARKER NO. 5) then [(MARKER NO. 5>=MARKER NO. 4)] else [(MARKER NO. 3<=MARKER NO. 2) nor (not(MARKER NO. 4<MARKER NO. 1))]

If (MARKER NO. 1=MARKER NO. 5) then [not(MARKER NO. 4>MARKER NO. 1)] else [(MARKER NO. 4>MARKER NO. 1) nor (MARKER NO. 2>−MARKER NO. 3)]

If (MARKER NO. 1=MARKER NO. 5) then [not(MARKER NO. 1<MARKER NO. 4)] else [(MARKER NO. 3=MARKER NO. 2) nor (MARKER NO. 1<MARKER NO. 4)]

Preferably, the assay kit of the invention comprises software, which is programmed to comprise the above logic rules, and which can—on basis of the stochastic values 0 or 1, resulting from the hybridization test—immediately yield the result of the test, i.e. whether the tested sample contains a pathogenic Legionella strain or not.

EXAMPLES DNA Isolation of Sample

In the present Example a total of 144 samples from different strains were selected from the collection of strains of the Streeklaboratorium Kennemerland in Haarlem, The Netherlands. Of these 144 samples, a total of 74 samples was isolated from hospital patients (pathogenic or clinical strains) and a total of 70 samples was isolated from industrial and public water supply systems, and were not detected in humans (environmental strains).

Labeling and Hybridization of Genomic DNA

All strains were cultivated after which genomic DNA was isolated from these strains. The DNA was then randomly (primer) labeled using Klenow DNA polymerase (BioPrime kit Invitrogen) according to the manufacturer's instructions.

Cy3 labeled marker sequences were prepared, comprising the sequences 1a, 2a, 3a, 4a and 5a. The DNA sample consisted of the DNA of the test strain and was labeled with Cy5. Both labeled samples were hybridized simultaneously to a microarray. Upon scanning and image analysis, the ratio of the Cy5/Cy3 emission values was calculated for each spot on the microarray. These ratio's served as data input for further data analysis.

All tests showed a positive control, i.e. the presence of L. pneumophila DNA was detected. Then, on basis of either positive or negative hybridization signals on the two markers (MARKER NO. 1 and MARKER NO. 2) or the five markers (MARKER NO. 1 through MARKER NO. 5) a classification of the sample as ‘pathogenic’ or ‘non-pathogenic’, respectively, was generated according to the rules as shown above. For the results, the sensitivity and specificity of the tests were calculated.

Sensitivity is calculated as the number of true positive classifications divided by the sum of the number of true positives and the number of false negatives as expressed by equation 1:

sensitivity=TP/(TP=FN)   (I)

wherein true positives are the number of pathogenic strains correctly identified and false negatives are the number of pathogenic strains incorrectly classified as being environmental strains.

Specificity is defined as the number of true negative results divided by the sum of the true negatives and the false positives as expressed by equation 2:

specificity=TN/(TN+FP)   (II)

wherein true negatives are defined as the number of environmental strains that are correctly identified and false positives are defined as the number of environmental strains that are identified as being pathogenic.

Array size Sensitivity (%) Specificity (%) 5 markers 95.8 61.4 2 markers 86.3 68.6 

1. A hybridization assay kit for use in a method to determine whether a Legionella pneumophila strain is a pathogenic strain or a non-pathogenic strain in a sample containing Legionella pneumophila, which comprises an array of less than 50 nucleotide sequences and which comprises a nucleotide sequence comprising at least 50 contiguous nucleotides from MARKER NO. 1 and a nucleotide sequence comprising at least 50 contiguous nucleotides from MARKER NO. 2 or homologues thereof.
 2. A hybridization assay kit according to claim 1, which further comprises nucleotide sequences comprising at least 50 contiguous nucleotides from each of MARKER NO. 3, MARKER NO. 4 and MARKER NO. 5 or homologues thereof.
 3. A hybridization assay kit according to claim 1, which further comprises a positive control sequence.
 4. A hybridization assay kit according to claim 1, which further comprises software for calculating the results of the logic rule If (MARKER NO. 1>MARKER NO. 2) then said strain is pathogenic wherein MARKER NO. 1 and MARKER NO. 2 are stochastic values representing 1 if hybridization and 0 if no hybridization has been observed with said sequences.
 5. A hybridization assay kit according to claim 2, which further comprises software for calculating the results of the logic rule (1) If (MARKER NO. 2<MARKER NO. 1) then [not(MARKER NO. 2=MARKER NO. 3)] else [(MARKER NO. 2=MARKER NO. 4) and (MARKER NO. 5=MARKER NO. 3)] (2) If (MARKER NO. 2<MARKER NO. 3) then [not(MARKER NO. 1<MARKER NO. 4)] else [(MARKER NO. 5>MARKER NO. 2) nor (MARKER NO. 5<MARKER NO. 4)](3) If (MARKER NO. 2<MARKER NO. 3) then [not(MARKER NO. 4<MARKER NO. 1)] else [(MARKER NO. 3<MARKER NO. 5) nor (MARKER NO. 4>MARKER NO. 5)] (4) If (MARKER NO. 1=MARKER NO. 5) then [(MARKER NO. 4<=MARKER NO. 1)] else [(MARKER NO. 1<MARKER NO. 4) nor (MARKER NO. 3=MARKER NO. 2)] (5) If (MARKER NO. 1=MARKER NO. 5) then [ (MARKER NO. 5>=MARKER NO. 4)] else [(MARKER NO. 3<=MARKER NO. 2) nor (not(MARKER NO. 4<MARKER NO. 1))] (6) If (MARKER NO. 1=MARKER NO. 5) then [not(MARKER NO. 4>MARKER NO. 1)] else [(MARKER NO. 4>MARKER NO. 1) nor (MARKER NO. 2>=MARKER NO. 3)] (7) If (MARKER NO. 1=MARKER NO. 5) then [not(MARKER NO. 1<MARKER NO. 4)] else [(MARKER NO. 3=MARKER NO. 2) nor (MARKER NO. 1<MARKER NO. 4)], wherein MARKER NO. n (n=1-5) is the stochastic value representing 1 if hybridization and 0 if no hybridization has been observed with said sequence n, and wherein said strain is pathogenic if 4 or more of the logic rules (1)-(7) have the result TRUE.
 6. A hybridization assay kit according to claim 3, wherein the positive control sequence is Legionella 30S ribosomal protein S21 or a conserved sequence of one of the Legionella housekeeping enzymes. 